Antibodies against biotin and uses thereof

Abstract

The application discloses an anti-biotin antibody and application thereof, wherein the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2 and CDR3 of the antibody are respectively shown as SEQ ID NO. 1-3; the amino acid sequences of the light chain complementary determining regions CDR1, CDR2 and CDR3 are respectively shown as SEQ ID NO. 4-6. The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8. The application enhances the affinity and stability of the antibody by engineering treatment, computer scanning and improvement of the CDR sequence space, screens the most reasonable binding conformation, and obtains a specific binding site of the biotin L-biotin specific binding antibody.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and in particular to an anti-biotin antibody and its applications. Background Technology

[0002] Biotin, also known as vitamin H or vitamin B7, is a water-soluble vitamin belonging to the B vitamin group. Its main function in the human body is as an essential coenzyme in the metabolism of carbohydrates, fats, and proteins within cells. Biotin is also crucial for maintaining healthy skin, hair, and the nervous system. Biotin exists in two forms: levorotatory L-B7 and dextrorotatory D-B7. Synthetic biotin is a racemic product.

[0003] The biotin-avidin system (BAS) refers to the extremely strong non-covalent interaction between biotin and avidin, exhibiting exceptionally high affinity. This makes the system widely used in biochemical and medical research. This binding characteristic makes the BAS a key component in many biomolecular labeling, localization, and purification techniques.

[0004] However, when using the streptavidin-biotin system for immunoassay, the presence of high concentrations of free biotin in the sample can compete with biotinylated antibodies for binding sites, thus affecting the test results. Anti-biotin antibodies have a strong specific binding affinity to biotinylated antibodies but a weaker binding affinity to free biotin. They can serve as a substitute for streptavidin, specifically binding to biotinylated antibodies and mitigating interference from free biotin in the sample to some extent. Currently, there are few biotin-reducing agents on the market, and their specificity is not ideal. Therefore, developing high-quality, highly specific biotin-reducing agents is essential. Summary of the Invention

[0005] The purpose of this invention is to provide a highly specific anti-biotin antibody that binds to L-biotin and avoids interference from endogenous D-biotin.

[0006] Biotin-binding antibody or its antigen-binding fragment, wherein the amino acid sequences of the heavy chain complementarity-determining regions CDR1, CDR2, and CDR3 of the antibody or its antigen-binding fragment are shown in SEQ ID NO. 1-3;

[0007] SEQ ID NO.1: SAGVT

[0008] SEQ ID NO.2:TINFAGTTYYNPALKS

[0009] SEQ ID NO.3: DLYSADGDPYWHLDY

[0010] The amino acid sequences of the light chain complementarity-determining regions CDR1, CDR2, and CDR3 of the antibody or its antigen-binding fragment are shown in SEQ ID NO. 4-6.

[0011] SEQ ID NO.4: SGSSSNIGRLGVT

[0012] SEQ ID NO.5: GTNKRPS

[0013] SEQ ID NO.6: AAVDWYMMGGV

[0014] The antibodies or their antigen-binding fragments described above can specifically bind to biotin and have high affinity. They have a strong specific binding ability with biotinylated antibodies, but a weaker binding ability with free biotin, thus eliminating interference caused by free biotin in the sample.

[0015] The amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is as shown in SEQ ID NO. 7 or has at least 80% similarity to the amino acid sequence shown in SEQ ID NO. 7.

[0016] SEQ ID NO.7: QVQLQESGPSLVKPSQTLSLTCTVSGFSLSSAGVTWVRQAPGKALEWVTTINFAGTTYYNPALKSRLSITRDTSKSQVSLSLSSVTIEDTAVYYCARDLYSADGDPYWHLDYWGPGLLATVSS

[0017] The amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment is as shown in SEQ ID NO. 8 or has at least 80% similarity to the amino acid sequence shown in SEQ ID NO. 8.

[0018] SEQ ID NO.8: QVVLTQPPAVSGSLGQTVTISCSGSSSNIGRLGVTWYQQLPGSAPRSLIYGTNKRPSGVPDRFSGTKSGNTGTLIITSLQPEDEADYYCAAVDWYMMGGVFGSGTRLTVL

[0019] In the case of having the above-described heavy chain complementarity-determining regions CDR1, CDR2, CDR3 and light chain complementarity-determining regions CDR1, CDR2, CDR3, the amino acid sequence of the heavy chain variable region having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID NO. 7, and the amino acid sequence of the light chain variable region having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID NO. 8, the antibody or its antigen-binding fragment corresponding to the amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence similarity, is also within the scope of protection of this invention.

[0020] In this invention, the antigen-binding fragment refers to a polypeptide containing a partial sequence fragment of the antibody light chain variable region and / or heavy chain variable region, which retains the ability to specifically bind biotin, or can compete with the full-length antibody for specific binding to biotin.

[0021] The antibody or its antigen-binding fragment is selected from any one of monoclonal antibodies, Fab, Fab', F(ab)2, Fd, Fv, dAb, complementarity-determining region fragments, and single-chain antibodies.

[0022] Bispecific or multispecific antibodies comprising the biotin-binding antibody or its antigen-binding fragment.

[0023] A nucleic acid molecule that encodes the above-described biotin-binding antibody or its antigen-binding fragment;

[0024] Based on the amino acid sequence and codon rules of the antibody or its antigen-binding fragment described above, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the above-described antibody or its antigen-binding fragment. Due to the degeneracy of codons, the nucleotide sequence encoding the above-described antibody or its antigen-binding fragment is not unique, and all nucleic acid molecules capable of encoding the above-described antibody or its antigen-binding fragment are within the scope of protection of this invention.

[0025] The biomaterial comprises the aforementioned nucleic acid molecule, and the biomaterial is any one of an expression cassette, a vector, or a host cell.

[0026] The antibody conjugate is obtained by conjugating the biotin-binding antibody or its antigen-binding fragment, or the bispecific antibody or multispecific antibody, to a label or protein; the label is selected from one or more of chemiluminescent dye labeling, enzyme labeling, biotin labeling, fluorescent dye labeling, colloidal gold labeling, and radioactive labeling.

[0027] The antibody or its antigen-binding fragment, the nucleic acid molecule, the biological material, the antibody conjugate, or the antibody composition may be used in any of the following ways:

[0028] (1) Application in the preparation of products for detecting the presence or level of biotin in a sample;

[0029] (2) Application in the preparation of products for diagnosing bacterial infections;

[0030] (3) Application in detecting the presence or level of biotin in a sample for non-diagnostic and therapeutic purposes;

[0031] (4) Use in the preparation of products for assessing the occurrence, development and / or prognosis of inflammation.

[0032] In the applications described in (1) and (2) above, the product may be a detection reagent or a kit. The sample described in (1) may be a sample from a living human or animal (including blood, etc.), or it may be a sample from a living human or animal, such as cells or cell culture medium cultured in vitro.

[0033] The samples mentioned in (3) above are not from living people or animals, such as: cells cultured in vitro, cell culture medium, etc.

[0034] After biotin labeling the target protein, an enzyme-labeled anti-biotin antibody is used to bind it, and protein expression is detected by a colorimetric reaction (such as HRP-catalyzed DAB colorimetric reaction).

[0035] Biotinylated primary or secondary antibodies bind to fluorescently labeled anti-biotin antibodies, enabling the localization and visualization of antigens in tissues or cells. For example, this can be used to detect the expression levels of specific proteins in tumor tissue sections.

[0036] By immobilizing anti-biotin antibodies on a chromatography column (such as agarose beads), biotinylated nucleic acids, proteins, or small molecules can be specifically captured, achieving high-purity separation.

[0037] In sandwich ELISA, biotinylated detection antibodies bind to enzyme-labeled anti-biotin antibodies, and the signal is amplified by substrate color development, thereby improving detection sensitivity.

[0038] Applications of biotinylated antibodies include, but are not limited to: ELISA detection, cell capture, sorting and enrichment, immunoblotting detection, flow cytometry detection, immunofluorescence staining, and immunohistochemical detection.

[0039] This invention utilizes computational antibody design to rationally engineer antibodies, enhancing their affinity and stability through computer scanning and modification of the CDR sequence space. In protein structure prediction, homology modeling is generally used to ensure accuracy. However, constructing precise protein structures solely based on amino acid sequences remains a challenge. To address this, in designing the antibody, we obtained a weakly biotin-binding Antibody-test1 antibody through experimental testing. We obtained the gene sequence of the Antibody1-test1 antibody using DNA sequencing technology and translated it to obtain its amino acid sequence. To enhance the affinity and stability of the Antibody1-test1 antibody, we theoretically designed amino acid mutations to obtain a more specific Antibody1 sequence. Finally, we expressed this sequence as the target antibody Antibody1 (e.g., ...) using experimental techniques. Figure 1 We obtained the gene sequence of the Antibody1-test1 antibody using DNA sequencing technology, translated it to obtain the amino acid sequence, and then used the online software SWISS-MODEL to model its three-dimensional structure. Protein modeling methods mainly include template modeling and de novo predictive modeling, among which homology modeling is the most widely used and its accuracy is relatively reliable. The online software SWISS-MODEL has a real-time supplemented protein database, and all protein structures of this invention were generated by this software, thus ensuring the reliability of the protein structures.

[0040] Using Neopharm's online tools, the CDR region of the antibody was defined according to the Kabat protocol for sequence variability.

[0041] In this implementation case, we constructed the complete protein structure of the antibody's light and heavy chains and determined the antibody's binding ability to biotin by changing the hole positions within the antibody. Based on this, we adopted the following implementation plan: First, we extracted the 3D protein structure generated by SWISS-MODEL software and saved it as a PDB file. Then, we imported it into the computational software Discovery Studio and used its Define and Edit Binding Site's Define Receptor function to define the antibody (Antibody.pdb) as the receptor molecule. We then used the Define and Edit Binding Site's From Receptor Cavities function to obtain 15 potential binding sites. Molecular docking was performed using biotin B7-L.pdb as the ligand and anti-Antibody.pdb as the receptor. We expanded the Top Hits parameter and set the Pose Cluster Radius to 0.5 to ensure the docking conformations were as diverse as possible. The remaining parameters were calculated using default settings to obtain the docking results, and interaction analysis was performed to ensure the antibody's ability to specifically bind biotin.

[0042] Beneficial effects:

[0043] This invention enhances the affinity and stability of antibodies by engineering them and performing computer scanning and improvement on the CDR sequence space, thereby screening out the most reasonable binding conformation and obtaining a highly specific binding site for biotin B7-L (L-biotin) specific binding antibody.

[0044] This invention uses computer simulation and analysis to predict the binding energy, binding region, and binding ratio of candidate antibodies and antigens, allowing for rapid modification and improvement to optimize antibody performance as needed. By screening for suitable binding conformations through computer simulation, biotin-B7-L specific binding antibodies with higher binding capacity are obtained. Because computer simulation provides accurate molecular structure information, it avoids extensive laboratory experiments and potential technical problems and errors, thus significantly shortening antibody design time, reducing experimental costs and risks, and improving R&D efficiency and success rate. Furthermore, computer-aided research makes antibody therapy research more efficient and precise, further promoting the development of antibody therapy. Attached Figure Description

[0045] Figure 1 Antibody1 structure predicted by SWISS-MODEL;

[0046] Figure 2 Antibody-test1 spatial location 7;

[0047] Figure 3 Antibody-test2 spatial site 8;

[0048] Figure 4 Antibody-test3 spatial site 7;

[0049] Figure 5 Antibody-test4 Spatial site 4;

[0050] Figure 6 Antibody-test5 spatial site 7;

[0051] Figure 7 Antibody-test6 Spatial site 6;

[0052] Figure 8 Antibody1-1 Spatial Site 1;

[0053] Figure 9 Antibody1 Spatial location 2;

[0054] Figure 10 Antibody1 spatial location 3;

[0055] Figure 11 Antibody1 spatial location 4;

[0056] Figure 12 Antibody1 spatial location 5;

[0057] Figure 13 Antibody1 spatial location 6;

[0058] Figure 14 Antibody1 spatial location 7;

[0059] Figure 15 Antibody1 spatial location 8;

[0060] Figure 16 Antibody1 spatial location 9;

[0061] Figure 17 Antibody1 spatial location 10;

[0062] Figure 18 Antibody1 spatial location 11;

[0063] Figure 19 Antibody1 spatial location 12;

[0064] Figure 20 Antibody1 spatial location 13;

[0065] Figure 21 Antibody1 spatial location 14;

[0066] Figure 22 Antibody1 spatial location 15;

[0067] Figure 23 Evaluation results of the binding performance of antibody1, antibody1-1 and antibody1-2 with the interfering free biotin;

[0068] Figure 24 To explain the impact of interfering substances in the sample on antibody detection;

[0069] Figure 25 Results of CA125 blood sample testing. Detailed Implementation Plan

[0070] Example 1

[0071] We experimentally obtained a biotin-binding Antibody-test1 antibody with a weak effect. We obtained the gene sequence of the Antibody1-test1 antibody using DNA sequencing technology and translated it to obtain the amino acid sequence. To enhance the affinity and stability of the Antibody1-test1 antibody, we theoretically designed amino acid mutations. Finally, we obtained the target antibody Antibody1 through experimental techniques. Taking Antibody1-test1 as an example, the specific operation is as follows: [Amino acid sequence:]

[0072] Antibody1-test1-Light: EVVLTQPPAVSGSLGQTVTISCTGSSSNIDRLWVTWYQQLPGSAPRSLIYGTNKRPSGVPDRFSGTKSGNTGTLIITSLQPEDEADYYCAAVDWYMMGGVFGSGTR LTVLGQPKSAPSVTLFPPSTEELSTNKATVVCLINDFYPGSVNVVWKADGSTINQNVKTTQASKQSNSKYAASSYLTLTGSEWKSKSSYTCEVTHEGSTVTKTVKPS

[0073] Antibody1-test1-Heavy: QVFLQESGPSLVKPSQTLSLTGTVSGFSLSSAGVTWVRQAPGKALEWVTTINFAGTTYYNPALKSRLSITRDTSKSQVSLSLSSVTIEDTAVYYCARDLYSADGDPYWHLDYWGPGLLATVSSASTTPPK VYPLTSCCGDTSSSIVTLGCLVSSYMPEPVTVTWNSGALTSGVHTFPAILQSSGLYSLSSVVTVPASTSGAQTFICNVAHPASSTKVDKRVEPGCPDPCKHCRCPPPELPGGPSVFIFPPKPKDTLTISG The TPEVTCVVVDVGQDDPEVQFSWFVDNVEVRTARTKPREEQFNSTFRVVSALPIQHQDWTGGKEFKCKVHNEGLPAPIVRTISRTKGQAREPQVYVLAPPQEELSKSTLSVTCLVTGFYPDYIAVEWQKNGQPESEDKYGTTTSQLDADGSYFLYSRLRVDKNSWQEGDTYACVVMHEALHNHYTQKSISKPPGK files are imported into the SWISS-MODEL software to generate the Antibody1-test1.pdb file with the protein's three-dimensional structure. The Antibody1-test1.pdb file and the pre-prepared L-biotin.pdb file are then imported into the Discovery Studio software.

[0074] Potential binding (hole) sites in the protein were identified. The protein structure was imported into the computational software DiscoveryStudio, and the Define Receptor function under its Define and Edit Binding Site was used to define the antibody Antibody1-test1.pdb as a receptor molecule. Then, using the FromReceptor Cavities function under the Define and Edit Binding Site, 15 potential binding sites were obtained. Molecular docking was performed in these holes using biotin B7-L.pdb as a ligand. The Top Hits parameter was expanded, and the Pose Cluster Radius was set to 0.5 to ensure the docking conformation diversity was maximized; the remaining parameters were calculated using default settings to obtain the result Antibody1-test1-B7-L.dsv. This process was repeated to obtain the Antibody1-test1-B7-L.dsv results for the 15 different binding sites. The antibody-antigen complex structure was calculated using Analyze Protein Interface to obtain the amino acid interactions between the antibody and antigen at the binding interface.

[0075] We conducted interaction analysis on stable antibody-antigen complexes, primarily studying the hydrophobic, hydrogen-bonding, and electrostatic interactions between ligands and receptors at the binding interface. These forces promote the binding of ligands and receptors in specific ways. Hydrophobic interactions refer to the energy effect generated when polar groups in a protein molecule aggregate and repel hydrophobic groups due to electrostatic and hydrogen-bonding forces. The hydrophobic interactions within a protein molecule, to a certain extent, determine its structural stability. Hydrogen bonds are weak interactions formed between an electron-deficient hydrogen atom and a highly electronegative atom or group of atoms. Hydrogen bonds provide the binding energy necessary to maintain the spatial stability of a protein and can also dissociate rapidly during protein metabolism, thus playing a crucial role in protein spatial structure. Electrostatic interactions refer to the various electrostatic attraction forces between dipoles, induced dipoles, and charged groups in a protein molecule. Electrostatic interactions are essential for maintaining protein structure and biological activity.

[0076] Using NewpBio's online tools, the Kabat protocol (Kabat definition protocol: based on sequence variability, the most widely used) defines the CDR regions of antibodies as follows: In the Light chain, CDR1: 23-35, CDR2: 51-57, CDR3: 90-100; In the Heavy chain, CDR1: 31-35, CDR2: 50-65, CDR3: 98-112.

[0077] In binding (empty) site 7 (e.g.) Figure 2 The binding ratio of the Antibody1-test1-CDR region to B7-L is 25%, and the interaction energy with B7-L is 29.6 kcal / mol. The specific binding regions are shown in Table 1.

[0078] Other binding (cavitation) sites showed that the binding ratio of the Antibody1-test1-CDR region to B7-L was 0%.

[0079] Example 2

[0080] In the implementation case Antibody1-test2, we kept the number and position of amino acids in the CDR region unchanged, and mutated some amino acids:

[0081] Antibody1-test2-Light:

[0082] EVVLTQPPAVSGSLGQTVTISCTDSSSNIGRLIVTWYQQLPGSAPRSLIYGTNKRPSGVPDRFSGTKSGNTGTLIITSLQPEDEADYYCAAVDWYMMGGVFGSGTR LTVLGQPKSAPSVTLFPPSTEELSTNKATVVCLINDFYPGSVNVVWKADGSTINQNVKTTQASKQSNSKYAASSYLTLTGSEWKSKSSYTCEVTHEGSTVTKTVKPS

[0083] Antibody1-test2-Heavy: QVFLQESGPSLVKPSQTLGLTGTVSGFSLSSAGVTWVRFAPGKALEWVTTINFAGTTYYNPAGKSRLSITRDTSKSQVSLSLSSVTIEDTAVYYCARDLYSADGDPYWHLDYW GPGLLATVSSASTTPPKVYPLTSCCGDTSSSIVTLGCLVSSYMPEPVTVTWNSGALTSGVHTFPAILQSSGLYSLSSVVTVPASTSGAQTFICNVAHPASSTKVDKRVEPGCPD PCKHCRCPPPELPGGPSVFIFPPPKPKDTLTISGTPEVTCVVVDVGQDDPEVQFSWFVDNVEVRTARTKPREEQFNSTFRVVSALPIQHQDWTGGKEFKCKVHNEGLPAPIVRT ISRTKGQAREPQVYVLAPPQEELSKSTLSVTCLVTGFYPDYIAVEWQKNGQPESEDKYGTTTSQLDADGSYFLYSRLRVDKNSWQEGDTYACVVMHEALHNHYTQKSISKPPGK

[0084] A three-dimensional structure was constructed in the same manner as in the implementation case Antibody-test1 and molecularly docked with B7-L. Experimental results: In binding (hole) sites 8 (such as... Figure 3 The binding ratio of the Antibody1-test2-CDR region to B7-L increased to 60%, and the interaction energy with B7-L was 28.5 kcal / mol. The specific binding regions are shown in Table 2.

[0085] Other binding (cavitation) sites showed that the binding ratio of the Antibody1-test2-CDR region to B7-L was 0%.

[0086] Example 3

[0087] In the implementation case Antibody1-test3, we kept the number and position of amino acids in the CDR region unchanged, and mutated some amino acids:

[0088] Antibody1-test3-Light:

[0089] EVVLTQPPAVSGSLGQTVTISCTGSSNIGRLGVTWYQQLPGSAPRSLIYGTNKRPSGVPDRFSGTKSGNTGTLIITSLQPEDEADYYCAAVDWYMMGGVFGSGTR LTVLGQPKSAPSVTLFPPSTEELSTNKATVVCLINDFYPGSVNVVWKADGSTINQNVKTTQASKQSNSKYAASSYLTLTGSEWKSKSSYTCEVTHEGSTVTKTVKPS

[0090] Antibody1-test3-Heavy:

[0091] QVFLQESGPSLVKPSQTLSLTGTVSGFSLSSAGVTWVRQAPGKALEWVTTINFAGTTYYNPALKSRLSITRDTSKSQVSLSLSSVTIEDTAVYYCARDLYSADGDPYWHLDYW GPGLLATVSSASTTPPKVYPLTSCCGDTSSSIVTLGCLVSSYMPEPVTVTWNSGALTSGVHTFPAILQSSGLYSLSSVVTVPASTSGAQTFICNVAHPASSTKVDKRVEPGCPD PCKHCRCPPPELPGGPSVFIFPPPKPKDTLTISGTPEVTCVVVDVGQDDPEVQFSWFVDNVEVRTARTKPREEQFNSTFRVVSALPIQHQDWTGGKEFKCKVHNEGLPAPIVRT ISRTKGQAREPQVYVLAPPQEELSKSTLSVTCLVTGFYPDYIAVEWQKNGQPESEDKYGTTTSQLDADGSYFLYSRLRVDKNSWQEGDTYACVVMHEALHNHYTQKSISKPPGK

[0092] A three-dimensional structure was constructed in the same manner as in the implementation case Antibody-test1 and molecularly docked with B7-L. Experimental results: In binding (hole) site 7 (e.g. Figure 4 The binding ratio of the Antibody1-test3-CDR region to B7-L increased to 60%, and the interaction energy with B7-L was 28.5 kcal / mol. The specific binding regions are shown in Table 3.

[0093] Other binding (cavitation) sites showed that the binding ratio of the Antibody1-test3-CDR region to B7-L was 0%.

[0094] Example 4

[0095] In the implementation case Antibody1-test4, we kept the number and position of amino acids in the CDR region unchanged, and mutated some amino acids:

[0096] Antibody1-test4-Light:

[0097] EVVLTQPPAVSGSLGQTVTISCTGSSNIGRLGVTWYQQLPGSAPRSLIYGTNKRPSGVPDRFSGTKSGNTGTLIITSLQPEDEADYYCAAVDWYMMGGVFGSGTRLT VLGQPKSAPSVTLFPPSTEELSTNKATVVCLINDFYPGSVNVVWKADGSTINQNVKTTQASKQSNSKYAASSYLTLTGSEWKSKSSYTCEVTHEGSTVTKTVKPSECS

[0098] Antibody1-test4-Heavy:

[0099] QVFLQESGPSLVKPSQTLSLTGTVSGFSLSSAGVTWVRQAPGKALEWVTTINFAGTTYYNPALKSRLSITRDTSKSQVSLSLSSVTIEDTAVYYCARDLYSADGDPYWHLDYW GPGLLATVSSASTTPPKVYPLTSCCGDTSSSIVTLGCLVSSYMPEPVTVTWNSGALTSGVHTFPAILQSSGLYSLSSVVTVPASTSGAQTFICNVAHPASSTKVDKRVEPGCPD PCKHCRCPPPELPGGPSVFIFPPPKPKDTLTISGTPEVTCVVVDVGQDDPEVQFSWFVDNVEVRTARTKPREEQFNSTFRVVSALPIQHQDWTGGKEFKCKVHNEGLPAPIVRT ISRTKGQAREPQVYVLAPPQEELSKSTLSVTCLVTGFYPDYIAVEWQKNGQPESEDKYGTTTSQLDADGSYFLYSRLRVDKNSWQEGDTYACVVMHEALHNHYTQKSISKPPGK

[0100] A three-dimensional structure was constructed in the same manner as in the implementation case Antibody-test1 and molecularly docked with B7-L. Experimental results: In binding (hole) site 4 (e.g. Figure 5 The binding ratio of the Antibody1-test4-CDR region to B7-L decreased to 20%, and the interaction energy with B7-L was -151.5 kcal / mol. Specific binding regions are shown in Table 4.

[0101] Other binding (cavitation) sites showed that the binding ratio of the Antibody1-test4-CDR region to B7-L was 0%.

[0102] Example 5

[0103] In the implementation case Antibody1-test5, we kept the number and position of amino acids in the CDR region unchanged, and mutated some amino acids:

[0104] Antibody1-test5-Light:

[0105] EVVLTQPPAVSGSLGQTVTISCTGSSNIGRLGVTWYQQLPGSAPRSLIYGTNKRPSGVPDRFSGTKSGNTGTLIITSLQPEDEADYYCAAVDWYMMGGVFGSGTR LTVLGQPKSAPSVTLFPPSTEELSTNKATVVCLINDFYPGSVNVVWKADGSTINQNVKTTQASKQSNSKYAASSYLTLTGSEWKSKSSYTCEVTHEGSTVTKTVKPS

[0106] Antibody1-test5-Heavy:

[0107] QVFLQESGPSLVKPSQTLSLTGTVSGFSLSSAGVTWVRQAPGKALEWVTTINFAGTTYYNPALKSRLSITRDTSKSQVSLSLSSVTIEDTAVYYCARDLYSADGDPYWHLDYW GPGLLATVSSASTTPPKVYPLTSCCGDTSSSIVTLGCLVSSYMPEPVTVTWNSGALTSGVHTFPAILQSSGLYSLSSVVTVPASTSGAQTFICNVAHPASSTKVDKRVEPGCPD PCKHCRCPPPELPGGPSVFIFPPPKPKDTLTISGTPEVTCVVVDVGQDDPEVQFSWFVDNVEVRTARTKPREEQFNSTFRVVSALPIQHQDWTGGKEFKCKVHNEGLPAPIVRT ISRTKGQAREPQVYVLAPPQEELSKSTLSVTCLVTGFYPDYIAVEWQKNGQPESEDKYGTTTSQLDADGSYFLYSRLRVDKNSWQEGDTYACVVMHEALHNHYTQKSISKPPGK

[0108] A three-dimensional structure was constructed in the same manner as in the implementation case Antibody-test1 and molecularly docked with B7-L. Experimental results: In binding (hole) site 7 (e.g. Figure 6 The binding ratio of the Antibody1-test5-CDR region to B7-L increased to 38%, and the interaction energy with B7-L was -235.1 kcal / mol. The specific binding regions are shown in Table 5.

[0109] Other binding (cavitation) sites showed that the binding ratio of the Antibody1-test5-CDR region to B7-L was 0%.

[0110] Example 6

[0111] In the implementation case Antibody1-test6, we kept the number and position of amino acids in the CDR region unchanged, and mutated some amino acids:

[0112] Antibody1-test6-Light:

[0113] EVVLTQPPAVSGSLGQTVTISCTGSSNISRLDVTWYQQLPGSAPRSLIYGTNKRPSGVPDRFSGTKSGNTGTLIITSLQPEDEADYYCAAVDWYMMGGVFGSGTR LTVLGQPKSAPSVTLFPPSTEELSTNKATVVCLINDFYPGSVNVVWKADGSTINQNVKTTQASKQSNSKYAASSYLTLTGSEWKSKSSYTCEVTHEGSTVTKTVKPS

[0114] Antibody1-test6-Heavy:

[0115] QVFLQESGPSLVKPSQTLSLTGTVSGFSLSSAGVTWVRQAPGKALEWVTTINFAGTTYYNPALKSRLSITRDTSKSQVSLSLSSVTIEDTAVYYCARDLYSADGDPYWHLDYW GPGLLATVSSASTTPPKVYPLTSCCGDTSSSIVTLGCLVSSYMPEPVTVTWNSGALTSGVHTFPAILQSSGLYSLSSVVTVPASTSGAQTFICNVAHPASSTKVDKRVEPGCPD PCKHCRCPPPELPGGPSVFIFPPPKPKDTLTISGTPEVTCVVVDVGQDDPEVQFSWFVDNVEVRTARTKPREEQFNSTFRVVSALPIQHQDWTGGKEFKCKVHNEGLPAPIVRT ISRTKGQAREPQVYVLAPPQEELSKSTLSVTCLVTGFYPDYIAVEWQKNGQPESEDKYGTTTSQLDADGSYFLYSRLRVDKNSWQEGDTYACVVMHEALHNHYTQKSISKPPGK

[0116] A three-dimensional structure was constructed in the same manner as in the implementation case Antibody-test1 and molecularly docked with B7-L. Experimental results: In binding (hole) site 6 (e.g. Figure 7 The binding ratio of the Antibody1-test6-CDR region to B7-L was increased to 50%, and the interaction energy with B7-L was -199.5 kcal / mol. The specific binding regions are shown in Table 6.

[0117] Other binding (cavitation) sites showed that the binding ratio of the Antibody1-test6-CDR region to B7-L was 0%.

[0118] Example 7

[0119] In the implementation case Antibody1, we kept the number and position of amino acids in the CDR region unchanged, and mutated some amino acids:

[0120] Antibody1-Light:

[0121] QVVLTQPPAVSGSLGQTVTISCSGSSSNIGRLGVTWYQQLPGSAPRSLIYGTNKRPSGVPDRFSGTKSGNTGTLIITSLQPEDEADYYCAAVDWYMMGGVFGSGTRLT VLGQPKSAPSVTLFPPSTEELSTNKATVVCLINDFYPGSVNVVWKADGSTINQNVKTTQASKQSNSKYAASSYLTLTGSEWKSKSSYTCEVTHEGSTVTKTVKPSECS

[0122] Antibody1-Heavy:

[0123] QVQLQESGPSLVKPSQTLSLTCTVSGFSLSSAGVTWVRQAPGKALEWVTTINFAGTTYYNPALKSRLSITRDTSKSQVSLSLSSVTIEDTAVYYCARDLYSADGDPYWHLDYW GPGLLATVSSASTTPPKVYPLTSCCGDTSSSIVTLGCLVSSYMPEPVTVTWNSGALTSGVHTFPAILQSSGLYSLSSVVTVPASTSGAQTFICNVAHPASSTKVDKRVEPGCPD PCKHCRCPPPELPGGPSVFIFPPPKPKDTLTISGTPEVTCVVVDVGQDDPEVQFSWFVDNVEVRTARTKPREEQFNSTFRVVSALPIQHQDWTGGKEFKCKVHNEGLPAPIVRT ISRTKGQAREPQVYVLAPPQEELSKSTLSVTCLVTGFYPDYIAVEWQKNGQPESEDKYGTTTSQLDADGSYFLYSRLRVDKNSWQEGDTYACVVMHEALHNHYTQKSISKPPGK

[0124] A three-dimensional structure was constructed in the same manner as in the implementation case Antibody-test1, and molecular docking was performed with B7-L. The experimental results are as follows:

[0125] In binding (cavitation) site 1 (e.g.) Figure 8 The binding ratio of the Antibody1CDR region to B7-L is 33.3%, and the interaction energy with B7-L is 21.5 kcal / mol. The specific binding regions are shown in Table 7.

[0126] In binding (cavitation) site 2 (e.g.) Figure 9 The Antibody1CDR region binds to B7-L at a rate of 33.3%, and the interaction energy with B7-L is -153.2 kcal / mol. The specific binding regions are shown in Table 8.

[0127] In binding (cavitation) site 3 (e.g.) Figure 10 Antibody1 and B7-L have no binding region.

[0128] In binding (cavitation) site 4 (e.g.) Figure 11 The binding ratio of the Antibody1CDR region to B7-L is 0%, and the interaction energy with B7-L is 20.7 kcal / mol. The specific binding regions are shown in Table 9.

[0129] In binding (empty) site 5 (e.g.) Figure 12 Antibody1 and B7-L have no binding region.

[0130] In binding (cavitation) site 6 (e.g.) Figure 13 The binding ratio of the Antibody1CDR region to B7-L is 33.3%, and the interaction energy with B7-L is -379.8 kcal / mol. The specific binding regions are shown in Table 10.

[0131] In binding (empty) site 7 (e.g.) Figure 14 The Antibody1CDR region binds to B7-L at a rate of 100%, and the interaction energy with B7-L is -726.6 kcal / mol. The specific binding regions are shown in Table 11.

[0132] In binding (cavitation) site 8 (e.g.) Figure 15 The binding ratio of the Antibody1CDR region to B7-L is 0%, and the interaction energy with B7-L is -738.5 kcal / mol. The specific binding regions are shown in Table 12.

[0133] In binding (empty) site 9 (e.g.) Figure 16 Antibody1-1 and B7-L have no binding region.

[0134] In the binding (cavitation) site 10 (e.g.) Figure 17The binding ratio of the Antibody1CDR region to B7-L is 0%, and the interaction energy with B7-L is 18.6 kcal / mol. The specific binding regions are shown in Table 13.

[0135] In binding (cavitation) site 11 (e.g.) Figure 18 The binding ratio of the Antibody1CDR region to B7-L is 0%, and the interaction energy with B7-L is 11.8 kcal / mol. The specific binding regions are shown in Table 14.

[0136] In binding (cavitation) site 12 (e.g.) Figure 19 The binding ratio of the Antibody1CDR region to B7-L is 0%, and the interaction energy with B7-L is -471.5 kcal / mol. Specific binding regions are shown in Table 15.

[0137] In binding (cavitation) site 13 (e.g.) Figure 20 Antibody1 and B7-L have no binding region.

[0138] In binding (cavitation) site 14 (e.g.) Figure 21 The binding ratio of the Antibody1CDR region to B7-L is 0%, and the interaction energy with B7-L is 15.9 kcal / mol. Specific binding regions are shown in Table 16.

[0139] In binding (cavitation) site 15 (e.g.) Figure 22 Antibody1 and B7-L have no binding region.

[0140] Antibody1 maintains both the binding ratio of the CDR region to B7-L, preserving the antibody's specific binding ability, and possesses high binding strength, ensuring the effectiveness of antibody binding to the antigen. We expressed it, and the complete results are as follows.

[0141] The structure of the Antibody1-binding antibody is shown in the table below:

[0142] Light-Region SequenceFragment Residues Length LFR1 QVVLTQPPAVSGSLGQTVTISC 1-22 22 CDR-L1 SGSSSNIGRLGVT 23-35 13 LFR2 WYQQLPGSAPRSLIY 36-50 15 CDR-L2 GTNKRPS 51-57 7 LFR3 GVPDRFSGTKSGNTGTLIITSLQPEDEADYYC 58-89 32 CDR-L3 AAVDWYMMGGV 90-100 11 LFR4 FGSGTRLTVL 101-110 10 Tail GQPKSAPSVTLFPPSTEELSTNKATVVCLINDFYPGSVNVVWKADGSTINQNVKTTQASKQSNSKYAASSYLTLTGSEWKSKSSYTCEVTHEGSTVTKTVKPSECS 111-216 106 All QVVLTQPPAVSGSLGQTVTISCSGSSSNIGRLGVTWYQQLPGSAPRSLIYGTNKRPSGVPDRFSGTKSGNTGTLIITSLQPEDEADYYCAAVDWYMMGGVFGSGTRLT VLGQPKSAPSVTLFPPSTEELSTNKATVVCLINDFYPGSVNVVWKADGSTINQNVKTTQASKQSNSKYAASSYLTLTGSEWKSKSSYTCEVTHEGSTVTKTVKPSECS 1-216 216

[0143] Heavy-Region SequenceFragment Residues Length HFR1 QVQLQESGPSLVKPSQTLSLTCTVSGFSLS 1-30 30 CDR-H1 SAGVT 31-35 5 HFR2 WVRQAPGKALEWVT 36-49 14 CDR-H2 TINFAGTTYYNPALKS 50-65 16 HFR3 RLSITRDTSKSQVSLSLSSVTIEDTAVYYCAR 66-97 32 CDR-H3 DLYSADGDPYWHLDY 98-112 15 HFR4 WGPGLLATVSS 113-123 11 Tail ASTTPPKVYPLTSCCGDTSSSIVTLGCLVSSYMPEPVTVTWNSGALTSGVHTFPAILQSSGLYSLSSVVTVPASTSGAQTFICNVAHPASSTKVDKRVEPGCPDPCKHCRCPPPELPGGPSVFIFPPKPKDTLTISGTPEVTCVVVDVGQDDPEVQFSWFVDNVE VRTARTKPREEQFNSTFRVVSALPIQHQDWTGGKEFKCKVHNEGLPAPIVRTISRTKGQAREPQVYVLAPPQEELSKSTLSVTCLVTGFYPDYIAVEWQKNGQPESEDKYGTTTSQLDADGSYFLYSRLRVDKNSWQEGDTYACVVMHEALHNHYTQKSISKPPGK 124-454 331 All QVQLQESGPSLVKPSQTLSLTCTVSGFSLSSAGVTWVRQAPGKALEWVTTINFAGTTYYNPALKSRLSITRDTSKSQVSLSLSSVTIEDTAVYYCARDLYSADGDPYWHLDYW GPGLLATVSSASTTPPKVYPLTSCCGDTSSSIVTLGCLVSSYMPEPVTVTWNSGALTSGVHTFPAILQSSGLYSLSSVVTVPASTSGAQTFICNVAHPASSTKVDKRVEPGCPD PCKHCRCPPPELPGGPSVFIFPPPKPKDTLTISGTPEVTCVVVDVGQDDPEVQFSWFVDNVEVRTARTKPREEQFNSTFRVVSALPIQHQDWTGGKEFKCKVHNEGLPAPIVRT ISRTKGQAREPQVYVLAPPQEELSKSTLSVTCLVTGFYPDYIAVEWQKNGQPESEDKYGTTTSQLDADGSYFLYSRLRVDKNSWQEGDTYACVVMHEALHNHYTQKSISKPPGK 1-454 454

[0144] Example 9 Antibody performance evaluation

[0145] Using plate-based chemiluminescence immunoassay, antibodies Antibody1, Antibody1-test1, and Antibody1-test2 were evaluated in the CA125 project to compare the potency differences among the raw materials.

[0146] Evaluation information

[0147] 1.1 Coating Material

[0148] Information on coating materials

[0149] Material Name Original concentration Use concentration Antibody1 5mg / mL 2ug / ml Antibody1-test1 5mg / mL 2ug / mL Antibody1-test2 5mg / mL 2ug / mL

[0150] 1.2 Marking Materials

[0151] Marking material information

[0152] Material Name Original concentration Use concentration Labeled antibodies CA125 (M5807, M5814) (purchased from Nanjing Jingda Biotechnology Co., Ltd.) 5mg / ml 0.8ug / ml

[0153] 1.3 Evaluation Process

[0154] Antibody1, Antibody1-test1, and Antibody1-test2 were coated with 2 μg / ml of each. Different dilutions of CA125 antigen, L-type biotinylate primary antibody (0.8 μg / ml), and HRP secondary antibody (1 / 8W) were added, 50 μl of each. The plates were incubated at 37℃ for 1 h. After washing and color development, the results were read as follows: Figure 23-25 As shown.

[0155] Figure 23 The middle part is the detection of the degree of binding between antibody1, antibody1-1, and antibody1-2 and the interfering free biotin; the lower the OD value, the better the antibody.

[0156] Figure 24 The value indicates the influence of interfering substances in the sample on antibody detection. The higher the value, the stronger the antibody's anti-interference ability and the better its performance, because interfering substances will reduce the detection value.

[0157] Figure 25 To test the CA125 blood sample results, the influence of interfering substances in the sample on antibody detection is investigated. The lower the value, the stronger the antibody's anti-interference ability and the better its performance, because interfering substances can cause some false positives.

[0158] 1.4 Evaluation Results

[0159] Experimental data:

[0160] As shown in Figure 23, at different antigen concentrations, Antibody1 showed a significantly higher titer than Antibody1-test1 (P < 0.01), while Antibody1-test2 showed a significantly higher titer (P < 0.05).

[0161] Experimental conclusion:

[0162] The test results showed that Antibody1 was more effective, significantly higher than Antibody1-test1 (P<0.01).

[0163] Table 1. Binding sites of Antibody-test1 and B7-L (7 sites)

[0164] Binding region residue sequence Combined with region type B:VAL2:HT3 - B7-L-1:O28 Hydrogen Bond B:PHE101:HN - B7-L-1:O12 Hydrogen Bond A:TRP47:HN - B7-L-1:O12 Hydrogen Bond B7-L-1:H10 - A:LEU45:O Hydrogen Bond B7-L-1:H30 - B:ASP93:OD2 Hydrogen Bond B:VAL100:HA - B7-L-1:O12 Hydrogen Bond B7-L-1:H31 - B:VAL2:O Hydrogen Bond B7-L-1:H32 - B:VAL2:O Hydrogen Bond

[0165] Table 2. Binding sites of Antibody-test2 and B7-L (8)

[0166] Binding region residue sequence Combined with region type B:VAL2:HT1 - B7-L-1:S11 Hydrogen Bond A:TRP47:HN - B7-L-1:O28 Hydrogen Bond A:ASN60:HD22 - B7-L-1:O29 Hydrogen Bond B7-L-1:H7 - A:ASN60:OD1 Hydrogen Bond B7-L-1:H7 - B7-L-1:O29 Hydrogen Bond B7-L-1:H10 - B:ASP93:OD2 Hydrogen Bond B7-L-1:H30 - B:GLY99:O Hydrogen Bond B:VAL100:HA - B7-L-1:O28 Hydrogen Bond A:GLU46:HA - B7-L-1:O28 Hydrogen Bond B7-L-1:H32 - A:ASN60:OD1 Hydrogen Bond

[0167] Table 3. Binding sites of Antibody-test3 and B7-L 7

[0168] Binding region residue sequence Combined with region type A:ARG71:HH21 - B7-L-1:O28 Hydrogen Bond B7-L-1:H30 - A:ASP72:O Hydrogen Bond A:ARG71:HD1 - B7-L-1:O12 Hydrogen Bond A:SER76:HA - B7-L-1:O28 Hydrogen Bond A:SER76:HB1 - B7-L-1:O28 Hydrogen Bond B7-L-1:H31 - A:ALA32:O Hydrogen Bond B7-L-1:H32 - A:ALA32:O Hydrogen Bond B7-L-1:S11 - A:PHE53 Other

[0169] Table 4. Binding sites of Antibody-test4 and B7-L.

[0170] Binding region residue sequence Combined with region type A:ARG71:HE - B7-L-1:O29 Hydrogen Bond B7-L-1:H7 - A:LEU29:O Hydrogen Bond B7-L-1:H10 - A:ASP33:OD1 Hydrogen Bond A:SER76:HA - B7-L-1:O29 Hydrogen Bond A:GLN77:HA - B7-L-1:O28 Hydrogen Bond

[0171] Table 5. Binding sites of Antibody-test5 and B7-L 7

[0172] Binding region residue sequence Combined with region type A:ARG71:HH21 - B7-L-1:O28 Hydrogen Bond B7-L-1:H30 - A:ASP72:O Hydrogen Bond A:ARG71:HD1 - B7-L-1:O12 Hydrogen Bond A:SER76:HA - B7-L-1:O28 Hydrogen Bond A:SER76:HB1 - B7-L-1:O28 Hydrogen Bond B7-L-1:H31 - A:ALA32:O Hydrogen Bond B7-L-1:H32 - A:ALA32:O Hydrogen Bond B7-L-1:S11 - A:PHE53 Other

[0173] Table 6. Binding sites of Antibody-test6 and B7-L.

[0174] Binding region residue sequence Combined with region type A:ARG71:HE - B7-L-1:O29 Hydrogen Bond A:ARG71:HD1 - B7-L-1:O12 Hydrogen Bond A:SER76:HB1 - B7-L-1:O28 Hydrogen Bond B7-L-1:H31 - A:ALA32:O Hydrogen Bond B7-L-1:H32 - A:ALA32:O Hydrogen Bond B7-L-1:S11 - A:PHE53 Other

[0175] Table 7. Antibody1 binding sites with B7-L 1

[0176] Binding region residue sequence Combined with region type B7-L-1:H7 - A:ASP86:OD2 Hydrogen Bond B7-L-1:H7 - A:TYR88:OH Hydrogen Bond B7-L-1:H10 - A:GLY104:O Hydrogen Bond B7-L-1:H30 - B:GLN39:OE1 Hydrogen Bond B:PRO41:HA - B7-L-1:O28 Hydrogen Bond B7-L-1:H31 - A:SER103:O Hydrogen Bond

[0177] Table 8. Antibody1 binding sites with B7-L 2

[0178] Binding region residue sequence Combined with region type B:GLY104:HN - B7-L-1:O28 Hydrogen Bond B7-L-1:H10 - B:ILE51:O Hydrogen Bond B7-L-1:H30 - B:GLY104:O Hydrogen Bond B7-L-1:H30 - B:ASP105:O Hydrogen Bond B:ILE51:HA - B7-L-1:O12 Hydrogen Bond B7-L-1:S11 - B:TRP108 Other

[0179] Table 9. Antibody1 binding sites with B7-L (4)

[0180] Binding region residue sequence Combined with region type A:LYS211:HN - B7-L-1:O12 Hydrogen Bond B:SER136:HG - B7-L-1:O28 Hydrogen Bond B:CYS137:HN - B7-L-1:O29 Hydrogen Bond B7-L-1:H10 - A:GLU214:OE1 Hydrogen Bond B:SER136:HA - B7-L-1:O29 Hydrogen Bond B:SER136:HB2 - B7-L-1:O28 Hydrogen Bond B7-L-1:H31 - B:CYS138:O Hydrogen Bond B7-L-1:H32 - B:CYS138:O Hydrogen Bond

[0181] Table 10 Antibody1 binding sites with B7-L 6

[0182] Binding region residue sequence Combined with region type B:ARG71:HH21 - B7-L-1:S11 Hydrogen Bond B:THR73:HG1 - B7-L-1:O28 Hydrogen Bond B:SER76:HN - B7-L-1:O29 Hydrogen Bond B7-L-1:H10 - B:ALA32:O Hydrogen Bond B7-L-1:H30 - B:SER74:O Hydrogen Bond B:SER74:HA - B7-L-1:O28 Hydrogen Bond B:SER76:HB1 - B7-L-1:O29 Hydrogen Bond B7-L-1:H31 - B:LEU29:O Hydrogen Bond B7-L-1:H31 - B:ALA32:O Hydrogen Bond

[0183] Table 11 Antibody1 and B7-L binding sites 7

[0184] Binding region residue sequence Combined with region type B:SER31:HG - B7-L-1:S11 Hydrogen Bond B:ALA32:HN - B7-L-1:S11 Hydrogen Bond B7-L-1:H10 - B:GLY33:O Hydrogen Bond B7-L-1:H31 - B:GLY33:O Hydrogen Bond B7-L-1:S11 - B:SER31:OG Other

[0185] Table 12 Antibody1 and B7-L binding sites (8)

[0186] Binding region residue sequence Combined with region type A:GLN39:HE22 - B7-L-1:O28 Hydrogen Bond B7-L-1:H7 - A:TYR37:O Hydrogen Bond A:GLN38:HA - B7-L-1:O12 Hydrogen Bond A:TYR87:HA - B7-L-1:O12 Hydrogen Bond B7-L-1:H31 - A:GLN39:OE1 Hydrogen Bond B7-L-1:H32 - A:GLN39:OE1 Hydrogen Bond

[0187] Table 13 Antibody1 and B7-L binding sites 10

[0188] Binding region residue sequence Combined with region type A:GLY146:HN - B7-L-1:O12 Hydrogen Bond A:LYS170:HZ2 - B7-L-1:O28 Hydrogen Bond A:TYR176:HH - B7-L-1:O28 Hydrogen Bond B7-L-1:H7 - A:GLU84:OE1 Hydrogen Bond B7-L-1:H7 - A:TYR176:OH Hydrogen Bond B7-L-1:H10 - A:GLY146:O Hydrogen Bond B7-L-1:H30 - A:GLU84:OE1 Hydrogen Bond A:GLY146:HA2 - B7-L-1:O12 Hydrogen Bond A:LYS170:HE1 - B7-L-1:O28 Hydrogen Bond

[0189] Table 14 Antibody1 binding sites with B7-L 11

[0190] Binding region residue sequence Combined with region type A:THR135:HG1 - B7-L-1:O12 Hydrogen Bond A:THR183:HG1 - B7-L-1:O12 Hydrogen Bond B:GLN181:HE21 - B7-L-1:O12 Hydrogen Bond B:GLN181:HE22 - B7-L-1:O12 Hydrogen Bond B:SER182:HN - B7-L-1:O28 Hydrogen Bond B:SER182:HG - B7-L-1:O28 Hydrogen Bond B7-L-1:H10 - A:GLU128:OE2 Hydrogen Bond

[0191] Table 15 Antibody1 binding sites with B7-L 12

[0192] Binding region residue sequence Combined with region type A:THR126:HG1 - B7-L-1:S11 Hydrogen Bond B:THR135:HN - B7-L-1:O29 Hydrogen Bond B7-L-1:H30 - A:PRO123:O Hydrogen Bond B7-L-1:H30 - B:THR135:O Hydrogen Bond A:SER125:HB1 - B7-L-1:O28 Hydrogen Bond A:SER125:HB2 - B7-L-1:O28 Hydrogen Bond B:LEU134:HA - B7-L-1:O29 Hydrogen Bond

[0193] Table 16 Antibody1 and B7-L binding sites 11

[0194] Binding region residue sequence Combined with region type A:ASN141:HD22 - B7-L-1:O29 Hydrogen Bond B7-L-1:H7 - A:ASP142:OD2 Hydrogen Bond B7-L-1:S11 - B:HIS174 Other

Claims

1. A biotin-binding antibody or its antigen-binding fragment, characterized in that, The amino acid sequences of the heavy chain complementarity-determining regions CDR1, CDR2, and CDR3 of the antibody or its antigen-binding fragment are shown in SEQ ID NO. 1-3, respectively; the amino acid sequences of the light chain complementarity-determining regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO. 4-6, respectively.

2. The biotin-binding antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is as shown in SEQ ID NO.7 or has at least 80% similarity to the amino acid sequence shown in SEQ ID NO.

7.

3. The biotin-binding antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment is as shown in SEQ ID NO.8 or has at least 80% similarity to the amino acid sequence shown in SEQ ID NO.

8.

4. A nucleic acid molecule, said nucleic acid molecule encoding the biotin-binding antibody or its antigen-binding fragment as described in any one of claims 1 to 3.

5. A biomaterial comprising the nucleic acid molecule of claim 4, wherein the biomaterial is any one of an expression cassette, a vector, or a host cell.

6. A test kit, characterized in that, Includes the biotin-binding antibody or its antigen-binding fragment as described in any one of claims 1 to 3.

7. The application of the biotin-binding antibody or its antigen-binding fragment as described in claim 1 in ELISA detection, cell capture, sorting and enrichment, immunoblotting detection, flow cytometry detection, immunofluorescence staining, and immunohistochemical detection for non-disease diagnostic purposes.

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